Yeast prions and Hsp70 chaperones

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

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Abstract

Organisms encode multiple Hsp70s to regulate abundance of Hsp70 in accordance with need in response to changing environments and to provide a range of distinct Hsp70 functions for carrying out many different tasks within cells and across cell types. We constructed a yeast system to evaluate Hsp70s from any source and have found that all of the functionally redundant essential yeast Hsp70s possess distinct activities. We also developed our system to investigate functions of the two Hsp90 paralogs. We are continuing to use this system to investigate how Hsp70s and Hsp90s within and across species influence propagation of amyloid in vivo or act in cellular PQC processes. The many ways that prion phenotypes change when activities or abundance of Hsp70s or their cochaperones are altered provides a sensitive way to investigate even subtle functional distinctions among highly redundant Hsp70s, and a useful approach to uncover the underlying mechanisms. The large number of co-chaperones that act on different steps of the Hsp70 reaction cycle can cooperate to provide both a broad range of function and fine tuning of Hsp70 activity to specify its proper functions in defined roles in cells. We discovered that Sis1, a J-protein regulator of Hsp70, protects cells from a latent toxicity of PSI+ prions, composed of the translation termination factor Sup35. PSI+ prions can be lethal to cells expressing Sis1 lacking either its non-essential substrate-binding function or its ability to interact with Hsp70. Our findings show that protection by Sis1 requires it to bind Sup35 and cooperate with Hsp70 in a way that moderates toxic depletion of the essential Sup35 protein into prion aggregates. Hsp70 cooperates with Hsp90 to promote the proper folding and functions of many "client" proteins that regulate various fundamental cellular processes. Altering abundance or function of Hsp70 and Hsp90 can lessen pathology in models of protein folding disorders, while in the same models reducing chaperone activity can cause or exacerbate pathology. These protein chaperones therefore are promising therapeutic candidates for amyloid and other protein folding disorders and they are being evaluated intensively as drug targets. We are studying functional interactions of Hsp70 and Hsp90 using yeast, human and disease organism proteins. We have identified specific sites on human Hsp90s that are important for regulating functional output of interactions with Hsp70 and other co-chaperones. We also identified a site on Hsp90 that revealed how subtle structural differences determine functional distinctions of the two highly homologous, but functionally distinct, human Hsp90s. We identified changes at additional sites on human Hsp90 that improve or inhibit its ability to function in vivo. We used a biophysical assay to monitor how these changes affect conformational dynamics of Hsp90. Together our findings connected how specific changes in the Hsp90 reaction cycle relate to its functions in vivo, and provide a framework for understanding the molecular mechanics needed to specify action of these Hsp90s under different physiological and environmental conditions. Our work toward understanding what underlies specificity in activities of functionally redundant Hsp70s and Hsp90s provide insight for new approaches to modify Hsp90 activity in specific ways. This understanding can help guide decisions about which Hsp70 or Hsp90-family members would be most useful for such applications, or point to potential problems that could arise due to differences in ways that different Hsp70 or Hsp90 paralogs respond to specific compounds. Overall our work provides insight into functions of protein quality control factors that can help guide strategies for using chaperones as targets for therapy in protein folding disorders.